Metabolism of phenacetin in the rat - University of …SouzaConradAgnello1984... · METABOLISM OF...

METABOLISM OF PHENACETIN IN THE RAT

CONRAD AGNELLO D'SOUZA

B.Pharm.

submitted in partial

fulfilment of the requirements for the degree of

Master of Pharmacy

UNIVERSITY OF TASMANIA

HOBART

SEPTEMBER 1983.

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THE UNIVERSITY

Of TASMANIA' LIBRARY

11L4 VI I

SUMMARY

The abuse of phenacetin-containing analgesic mixtures has been

linked epidemiologically with nephrotoxicity and carcinogenicity

in man.

In addition, clinical and histopathological tests after chronic

administration of phenacetin in man and animals have indicated

that it is nephrotoxic and carcinogenic. Furthermore, it has a

chemical similarity to other known carcinogenic arylamides.

However, the induction of toxicity with phenacetin remains a

controversial subject.

A number of chronic dosing studies with phenacetin have been

carried out to demonstrate the ability of the drug to induce

carcinogenesis and nephropathy. However, none have sought to

explain the reasons for the chronic nature of phenacetin

toxicity on the basis of the increased formation of toxic

metabolites after continued administration of the drug. In the

present chronic daily-dosing study with phenacetin in the rat,

the metabolism of the drug was monitored by analysing urine

samples at regular weekly intervals. Particular attention was

paid to the formation of N-hydroxyphenacetin, which has been

implicated in phenacetin carcinogenicity.

The metabolism of phenacetin was monitored in five groups of

Hooded Wistar rats. Each group was subjected to a different

treatment. The purpose was to elucidate the effects of the size

of the dose, duration of treatment, influence of commonly co-

administered drugs (aspirin, caffeine) and the influence of a

sulfation inhibitor (pentachlorophenol) on the metabolism of

phenace tin.

The metabolic trends indicated auto-induction of N-

hydroxylation, evidenced by the increased formation of N-

hydroxyphenacetin in all treatments. The induction was most

pronounced with the large dose of phenacetin and, significantly,

was prominent with the co-administration of aspirin at the lower

dose of phenacetin.

Paracetamol-sulfate was the major metabolite of phenacetin in

the rat, while paracetamol-glucuronide and free paracetamol were

the other products of the deethylation pathway of phenacetin.

The mercapturate and cysteinyl conjugates were not detected.

Pentachlorophenol, a known inhibitor of sulfation, did not block

sulfation completely. The partial suppression of sulfation with

pentachlorophenol resulted in the increased formation of 2-

hydroxy-p-phenetidine and yielded a larger fraction of unchanged

phenacetin in the urine.

The metabolism of p-phenetidine, a deacetylated metabolite of

phenacetin, was followed in freshly isolated rat hepatocytes.

The biotransformation of p-phenetidine to phenacetin and 2-

hydroxyphenetidine indicated that N-acetylation was significant

in the Hooded Wistar rat. However, N-acetylation and aromatic

hydroxylation did not fully account for the disappearance of p-

phenetidine from the in vitro system.

CONTENTS

CHAPTER 1 : INTRODUCTION

1.1. ANALGESIC ABUSE ASSOCIATED TOXICITY AND PHENACETIN 1

1.1.1. ANALGESIC-INDUCED NEPHROPATHY IN MAN 1

1.1.2. ANALGESIC-INDUCED CARCINOMA IN MAN 2

1.1.3. PHENACETIN-INDUCED TOXICITY IN MAN 3

1.1.4. PHENACETIN-INDUCED TOXICITY IN ANIMALS 3

1.1.5. TOXICITY-INDUCED BY OTHER ANALGESICS 4

1.2. METABOLITES OF PHENACETIN 6

1.3. BIOTRANSFORMATION OF PHENACETIN 8

1.3.1.1. PARACETAMOL :

METABOLITE-MEDIATED TOXICITY 8

1.3.1.2. PARACETAMOL :

POSTULATED REACTIVE METABOLITES 10

1.3.1.2.1. N-HYDROXYPARACETAMOL 10

1.3.1.2.2. 3,4-EPDXIDE-PARACETAMOL 11

1.3.1.2.3. N-ACETYL-p-BENZOQUINONEIMINE 11

1.3.1.3. p-AMINOPHENOL 12

1.3.1.4. CONJUGATES OF PARACETAMOL 13

1.3.2. p-PHENETIDINE 13

1.3.3. 2-HYDROXYPHENACETIN, 3-HYDROXYPHENACETIN,

2-HYDROXYPHENETIDINE 14

1.3.4. N-HYDROXY METABOLITES 15

1.3.4.1. N-HYDROXYPHENACETIN : A PROXIMAL CARCINOGEN 17

1.4. CHRONIC DOSING STUDIES

18

1.4.1. ALTERED METABOLISM WITH CHRONIC DOSING 18

iv

1.4.2. INFLUENCE OF DOSE SIZE, ADMINISTRATION ROUTE,

SPECIES VARIATION AND DURATION OF TREATMENT 19

1.4.3. INFLUENCE OF CO-ADMINISTERED DRUGS 21

1.4.4. INFLUENCE OF AGE AND SEX 22

1.4.5. INFLUENCE OF SULFATION INHIBITION 23

1.5. POSTULATED MECHANISMS OF PHENACETIN-INDUCED TOXICITY 24

1.6. SCOPE OF THE PRESENT IN VIVO CHRONIC DOSING STUDY 25

1.7. ELUCIDATION OF p-PHENETIDINE METABOLISM IN VITRO 26

1.8. SELECTION OF AN IN VITRO SYSTEM 27

1.9. AIMS OF THE PRESENT STUDY 28

CHAPTER 2 : EXPERIMENTAL

2.1. MATERIALS 29

2.1.1. SYNTHESIS OF p-NITROSOPHENETOLE 30

2.2. ANIMALS 31

2.3. IN VITRO STUDY 31

2.3.1. HEPATOCYTE ISOLATION 31

2.3.2. INCUBATION AND SAMPLE COLLECTION 33

2.4. ANALYSIS OF THE METABOLITES OF p-PHENETIDINE 34

2.4.1 DETECTION AND MEASUREMENT OF PHENACETIN,

PARACETAMOL, p-AMINOPHENOL AND p-PHENETIDINE 34

2.4.1.1. HYDROLYSED SAMPLES 34

2.4.1.2. UNHYDROLYSED SAMPLES 35

2.4.2. DETECTION AND MEASUREMENT OF

p-NITROSOPHENETOLE AND N-HYDROXYPHENETIDINE 35

2.4.3. DETECTION AND MEASUREMENT OF


2.5. IN VIVO STUDY 37

V

2.5.1.1. CHOICE OF DOSE 37

2.5.1.2. TIME AND ROUTE 37

2.5.1.3. FREQUENCY AND DURATION 37

2.5.1.4. FORMULATION 37

2.5.2.1. DRUG TREATMENTS 38

2.5.2.2. URINE COLLECTION 38

2.6. ANALYSIS OF THE METABOLITES OF PHENACETIN 40

2.6.1. DETECTION AND QUANTITATION OF

PHENACETIN, 2-HYDROXYPHENACETIN AND

3-HYDROXYPHENACETIN 40


PARACETAMOL AND ITS CONJUGATES 40



2.6.4. ASSAY FOR N-HYDROXYPHENACETIN 41

2.7. STATISTICAL ANALYSIS OF DATA 43

CHAPTER 3 : RESULTS AND DISCUSSION

3.1. METABOLITES OF p-PHENETIDINE IN VITRO 44

3.2. METABOLITES OF PHENACETIN IN VIVO 46

3.2.1. P500 TREATMENT 48

3.2.2. P50 TREATMENT 51

3.2.3. P50/A TREATMENT 54

3.2.4. P50/C TREATMENT 57 -

CONCLUSION 59

REFERENCES 60

v i

This thesis contains no material which has been accepted for the

award of any other degree or diploma in any University.

To the best of my knowledge and belief, this thesis contains no

material previously published or written by another person,

except when due reference is made in the text. of the thesis.

(C.A.D' OUZA)

vii

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to Dr. Stuart McLean for

his constant encouragement and guidance throughout the course of

this study.

The facilities at the School of Pharmacy were kindly extended to

me by Dr. Alan Polack.

I wish to thank Heather Galloway for drawing the figures and

Helen Lawler for secretarial assistance.

I would also like to thank Mr. Noel Davies and the staff of the

Central Science Laboratory, University of Tasmania, who helped

with the Gas Chromatography—Mass Spectrometry.

In particular, I wish to thank my parents for the support and

encouragement I have always received from them.

ABBREVIATIONS

Phenacetin

APAP-SULF Paracetamol-sulfate

APAP-GLUC Paracetamol-glucuronide

APAP-CYS Paracetamol-cysteine

APAP-GSH Paracetamol-glutathione

APAP •Paracetamol

NHAPAP N-Hydroxyparacetamol

3,4-E 3,4-Epoxide-paracetamol

NAQ N-Acetyl-p-benzoquinoneimine

PAP p-Aminophenol

NHP N-Hydroxyphenacetin

NMP N-Methoxyphenacetin

DNHP Deuterated N-hydroxyphenacetin

DNMP Deuterated N-methoxyphenacetin

2HP 2-Hydroxyphenacetin

2MP 2-Methoxyphenacetin

PN p-Phenetidine

NHPN N-Hydroxyphenetidine

2HPN 2-Hydroxyphenetidine

NOPN p-Nitrosophenetole

N-O-GLUC N-O-Glucuronide

N-O-SULF N-O-Sulfate

PCP Pentachlorophenol

AAMB N-Acetyl-4-aminomethyl benzoate

BAMB N-Butyry1-4-aminomethyl benzoate

18-0, 14-C Isotopic oxygen, carbon

Degrees centigrade

viii

CHAPTER 1. INTRODUCTION

1.1. ANALGESIC ABUSE-ASSOCIATED TOXICITY AND PHENACETIN

Phenacetin, a widely used antipyretic-analgesic, was first

introduced for the treatment of pain in 1887. It continued to be

extensively used, chiefly as a constituent in analgesic

mixtures, until recently (Flower et al., 1980). Analgesic

nephropathy. has been recognized as a frequent clinical,

radiological and autopsy finding in Scandinavia (Lindeneg et

al.,1959; Bengtsson,1962; Harvald,1963), Australia (McCutcheon,

1962; Jacobs and Morris,1962; Dawborn et al.,1966; Kincaid-

Smith,1969), Britain (Jacobs,1964; Sanerkin and Weaver, 1964),

United States of America (Reynolds and Edmondson,1963) and

Canada (Lakey,1961). .However, controversy persists with regard

to the involvement of phenacetin in the development of analgesic

nephropathy (Freeland,1975; Nanra,1976).

1.1.1. ANALGESIC-INDUCED NEPHROPATHY IN MAN

Analgesic abuse, now defined as an intake in excess of 1 g of an

analgesic per day, for one year, by Bengtsson et al. (1978), was

first identified as a problem in Sweden, with particular

reference to phenacetin, in 1918 (Grimlund,1963). However, it

was not until 1953 that Spuhler and Zollinger (1953) pointed out

that analgesic drugs could cause chronic renal disease. It has

since been an area of concern demanding intensive research

effort to explain the induction of toxicity by analgesics.

Dubach and co-workers conducted an epidemiological study of the

abuse of analgesics in a Swedish population, over a period of

ten years from 1968-1979, and concluded that heavy users of

analgesic mixtures over the course of a decade exhibited a

higher incidence of both abnormal kidney function and kidney-

related mortality, though the absolute incidences remained small

even among heavy users (Dubach et al.,1968; 1971; 1975; 1983).

Further evidence implicating analgesics in nephrotoxicity was

provided by the studies of Duggin (1977), Bengtsson et al.

(1978), Kincaid-Smith (1978), Bengtsson and Angervall (1979) and

Prescott (1966; 1982), who examined the relationship between

ingestion of analgesics and the development of renal disease.

Analgesic abuse-associated nephropathy, as the condition is

known and described today, is affected by addiction or abuse of

alcohol, cigarettes, barbiturates, hypnotics, tranquilizers and

laxatives (Prescott,1976; Kincaid-Smith,1978; Nanra et al.,

1978).

1.1.2. ANALGESIC-INDUCED CARCINOMA IN MAN

Phenacetin has also been implicated as a cause of carcinoma of

the renal pelvis. Hultengren et al. (1965) were the first to

suggest the association of analgesic abuse with carcinoma in

Sweden. They were followed by researchers in other countries

(Taylor,1972; Liu et al.,1972; Bengtsson et al.,1978; Lornoy et

al.,1979), who provided further evidence on the relationship

between analgesic abuse and tumors of the renal pelvis.

1.1.3. PHENACETIN-INDUCED TOXICITY IN MAN

The decline in the use of phenacetin is also attributed to the

methemoglobinemia, sulfhemoglobinemia and hemolytic anemia it

produces on chronic administration (Flower et al.,1980), as well

as nephropathy (Spuhler and Zollinger,1953) and 'neoplasia (Liu

et al.,1972). Although the adverse effects produced by

phenacetin are reversed in most instances on withdrawal of the

drug, they still remain an alarming testimony of drug-induced

pathophysiological disorders. The fact that phenacetin was the

one common ingredient in all combination analgesics responsible

for renal impairment (Koutsaimanis and de Wardener,1970), was

used to incriminate it as the compound responsible for the

increased incidence of tumors of the renal pelvis (Hultengren et

al.,1965; Bengtsson et al.,1968; Angervall et al.,1969;

Johansson et a1,1974; Johansson and Wahlqvist 1977) interstitial

nephritis and renal papillary necrosis (Nordenfelt and Ringertz,

1961). It was also seen that the withdrawal of phenacetin from

analgesic preparations in Sweden in 1961 resulted in fewer

deaths from renal failure among analgesic abusers in subsequent

years (Nordenfelt,1972), an observation that added credence to

the alleged toxicity of phenacetin. However, recent data

suggests that phenacetin may not be the only toxic analgeaic

(Sec. 1.1.5).

1.1.4. PHENACETIN-INDUCED TOXICITY IN ANIMALS

In earlier animal experiments, phenacetin failed to manifest any

noteworthy toxicity. Neither phenacetin nor most of its

metabolites were proven. to be significantly nephrotoxic in

animals (Calder et al.,1971). Only minimal renal papillary

necrosis has been produced in animals by administration of

phenacetin alone, even in large doses (Clausen,1964; Fordham et

al.,1965). Although renal pelvic tumors in laboratory animals

have not been encountered after long-term administration of

phenacetin, it must be borne in mind that several bladder

carcinogens exhibit species specificity, and failure to produce

carcinoma in animals is not sufficient proof of their safety in

man (Nery,1971a). For example, 2-naphthylamine, a known

carcinogen in man, does not produce malignancies in rats,

rabbits, cats and mice (Bonser et al.,1959). However, in recent

years phenacetin has been shown to cause hepatic necrosis in

hamsters (Mitchell et al.,1975; 1976; Nelson et al.,1978).

Chronic administration of phenacetin to rats in the diet induced

urothelial hyperplasia of the renal papillae, earduct tumors and

mammary adenocarcinomas (Johansson and Angerval1,1976), nasal

carcinomas and urinary tract tumors (Isaka et al.,1979) and had

a carcinogenic effect on most tissues (Johansson,1981). These

studies suggested a more general carcinogenic effect of

phenacetin in the rat. The rat therefore has been chosen as a

suitable animal model for the investigation of the metabolism of

phenacetin to putative toxic metabolites.

1.1.5. TOXICITY INDUCED BY OTHER ANALGESICS

It should be noted, however, that renal papillary necrosis has

been produced by aspirin, phenacetin and caffeine mixtures and

by aspirin alone, more readily than by phenacetin itself

(Abrahams et al.,1964; Saker and Kincaid-Smith,1969; Nanra and

Kincaid-Smith,1970; 1972a; 1973b; Nanra et al.,1970). Lesions

were seen to develop more frequently at low dose levels with

aspirin than with phenacetin (Axelsen,1976). The substantial

increase in excretion of renal tubular cells (a gauge of renal

damage) observed in healthy volunteers receiving aspirin,

compared with those receiving phenacetin, was taken as further

evidence of the greater nephrotoxic risk of aspirin (Prescott,

1965) in comparison to phenacetin. Cognisance must also be

taken of the renal papillary necrosis reportedly induced in

animals with other analgesics and nonsteroidal anti-inflammatory

drugs including phenazone (Nanra and Kincaid-Smith,1973a)

indomethacin and phenylbutazone (Nanra et al.,1970; Arnold et

al.,1974), amidopyrine (Brown and Hardy,1968), mefenamic acid

(Nanra et al.,1970) and the sole responsibility of phenacetin

for such toxicity diminished.

Although in no cases reported have patients consumed phenacetin

alone, the incrimination of phenacetin as the nephrotoxic and

carcinogenic entity in analgesic mixtures has been made on the

presumption that its metabolism is similar to that of known

carcinogenic amines (Miller and Miller,1966a; 1966b; Bengtsson

et al.,1978). The metabolism of phenacetin has been

investigated in several species, including rats, hamsters,

rabbits and human subjects (Smith and Griffiths,1976; Kuntzman

et al.,1977; Prescott,1980; Vaught et al.,1981). However, there_

remains the need to elucidate, in greater depth, its metabolism

after chronic administration.

1.2. METABOLITES OF PHENACETIN

Several studies have been carried out to identify the

metabolites of phenacetin ,since Brodie and Axelrod (1949) and

Smith and Williams (1949) independently found N-acetyl-p-

aminophenol (paracetamol,APAP) as the major and p-phenetidine

(PN) as the minor metabolite of phenacetin. Jagenburg and

Toczko (1964) isolated S-(1-acetamido-4-hydroxyphenyl)cysteine

as a urinary metabolite of phenacetin in man, while Klutch et

al. (1966) identified 2-hydroxyphenacetin in the urine of

humans, dogs and cats. Buch et al. (1966,1967) detected 2-

hydroxyphenetidine (2HPN), in human and rat urine and 3-

hydroxyphenacetin in the urine of rats given phenacetin.

N-hydroxyphenacetin (NHP), whose conjugates are postulated to be

potential carcinogens, was reportedly first detected in'the

urine of cats and dogs treated with phenacetin (Klutch et al.,

1966). It was further found in the urine of humans and dogs by

Klutch and Bordun (1960), but had not been indisputably

demonstrated to be a metabolite of phenacetin in vivo

(Weisburger and Weisburger, 1973; Hinson and Mitchell, 1976),

until 1981 in the rat (McLean et al., 1981). Kiese and Lenk

(1969) detected 4-ethoxyglycolanilide as a phenacetin metabolite

in the urine of rabbits. Nery (1971b) found N-acetyl-S-

ethylcysteine, acetamide and quinol to be metabolites of

phenacetin in the rat. Focella et al. (1972) identified 4-

hydroxy-3-methylthio-acetanilide, while Fischbach et al. (1977)

found N-[4-(2-hydroxyethoxy)pheny1]-acetamide in the urine of

rats and rabbits after administration of phenacetin.

4-Acetaminophenoxyacetic acid was detected as a new metabolite

of phenacetin by Dittman and Renner (1977) and 3-methylthio-4-

hydroxyacetanilide by Klutch et al. (1978).

Recently there has been considerable interest in the disposition

of phenacetin and its metabolites in animals and in man

(Prescott et al.,1968; Kampffmeyer,1974; Raaflaub and Dubach,

1975; Welch et al.,1976) with relevance to enzyme induction,

which increased the metabolism of phenacetin (Pantuck et al.,

1974). Habitual smoking is reported to enhance the metabolism

of phenacetin (Pantuck et al.,1972) and dietary habits such as

the consumption of charcoal broiled beef have been known to

increase the metabolism of phenacetin (Conney et al.,1976). The

potential interaction of reactive metabolites of phenacetin with

biological macromolecules has also been investigated in recent

years (Mulder et al.,1977; Hinson et al.,1977).

G LUC

SU LF

I HYDROXYLATION

0C2 H 5

NH P

0C2H5

DEETHYLATION

0064906

APAP-GLUC

NH2 NHCOCH3

OH

PAP

OH

APAP

NH2 CH2CH

‘ COOH

GLUC

SULF

DEACE TV LAT ION

9" NCOCH3

NHCOCH3

OH

0C2H5

3HP

GLUC

SULF

HYDROXYLATION •

G LUC

SU L F

0C2H5

PN

0C 2 H5

2H P

NH 2

OH

L--)

0C2 H5

2HPN

0C 2 H 5

NOPN

0C 2 H 5

NHPN

OSO3H

APAP-SULF

FIG. I: Biotransformation of phenacetin

1.3. BIOTRANSFORMATION OF PHENACETIN

The metabolic biotransformations by which phenacetin is

hydroxylated, oxidatively deethylated, N-deacetylated and

conjugated by hepatic microsomal enzymes (Brodie and Axelrod,

1949; Smith and Williams,1949; Klutch et a1,1966; Buch et al.,

1967; Prescott,1969; Nery,1971b; Focella et al.,1972; Mrochek

et al.,1974) are depicted in Fig. 1.

1.3.1.1. PARACETAMOL: METABOLITE-MEDIATED TOXICITY

Paracetamol is the major, immediate metabolite of phenacetin

(Brodie and Axelrod,1949) and is a widely used antipyretic-

analgesic in its own right. It is known to be hepatotoxic in

massive overdosage (Boyd and Bereczky,1966; Prescott et al.,

1971; Mitchell et al.,1973a; Kleinman et al.,1980) and

nephrotoxic following prolonged abuse (Duggin and Mudge,1976;

Mitchell et al.,1977; Mudge et al.,1978) due to metabolic

activation to a highly reactive toxic metabolite(Mitchell et

al.,1973a; 1973b; Jollow et al.,1973; Potter et al. 1973;

Hinson et al.,1980; Hinson and Gillette,1980).

The implication of a reactive metabolite in paracetamol-induced

toxicity was first disclosed by the studies of Mitchell et al.

(1973a), who showed that paracetamol produced an increased

incidence of hepatic necrosis in rats in which drug metabolizing

enzymes were induced by prior treatment with phenobarbitone or

3-methylcholanthrene, and that a decrease in the incidence and

severity of toxicity followed the use of inhibitors of drug

metabolism such as piperonyl butoxide or cobaltous chloride

(Potter et al., 1974) and recently, cimetidine (Mitchell et al.,

1981). This reactive metabolite is formed by a microsomal

cytochrome P-450 mixed function oxidase and is detoxified by

conjugation with glutathione (Mitchell et al.,1973b; Jollow et

al., 1973).

Paracetamol has also been recognized as a nephrotoxic metabolite

of phenacetin by other workers such as Nanra et al. (1980) and

Margetts (1976). Further observations that patients who

persisted in abusing analgesic mixtures in which phenacetin was

replaced with salicylamide or paracetamol still presented with

typical analgesic nephropathy and renal failure (Krishnaswamy

and Nanra,1976; Nanra et al.,1978) corroborates the nephrotoxic

potential of paracetamol.

In animal studies Nanra et al. (1970) and Nanra and Kincaid-

Smith (1970; 1972b) established a similarity between the

nephrotoxicity caused by phenacetin and that produced by

paracetamol when administered alone or in combination with other

analgesic constituents. It seems apparent, therefore, that if

the toxicity caused by the chronic ingestion of phenacetin alone

is accepted, then it is metabolite-mediated and could be at

least partly due to one or more of the highly reactive

metabolites of paracetamol (Sec. 1.3.1.2)

1.3.1.2. PARACETAMOL : POSTULATED REACTIVE METABOLITES

Jollow et al. (1973) and Mudge et al. (1978) established that

acute overdosage of paracetamol in rats and mice ultimately

resulted in covalent binding of the reactive metabolite to

tissue macromolecules in the liver and kidney, after the

depletion of glutathione. Although a study of urinary

metabolites has indicated that reactive intermediates had

reacted with cellular glutathione (Hinson et a1,1980; Jollow et

al.,1974a) none of the postulated toxic intermediates have been

identified as being solely responsible for the toxicity, during

the course of in vivo and in vitro (microsomal) experimentation.

1.3.1.2.1. N-HYDROXYPARACETAMOL

Among the proposed toxic metabolites (Fig. 2) was N-

hydroxyparacetamol (NHAPAP).This had been earlier shown to

dehydrate to N-acetyl-p-benzoquinoneimine (NAQ), a compound

known to react with cellular glutathione and protein and

postulated to be toxic (Jollow et al.,1974a). Therefore NHAPAP

was believed to be the toxic reactive intermediate of

paracetamol (Mitchell et al., 1973a; 1973b; Jollow et al.,1973;

Potter et al.,1973; 1974). This concept was questioned because

NHAPAP was not detected as a metabolite of paracetamol although

it is formed from phenacetin (Hinson et al.,1979a; Nelson et

al.,1980). At a physiological pH NHAPAP did not rapidly

dehydrate to NAQ (Healey et al.,1978; Gemborys et al.,1980) and

was only slightly more toxic than APAP (Healey et al.,1978),

hence could not be regarded as its toxic intermediate.

1.3.1.2.2. 3-4,EPDXIDE-PARACETAMOL

The implication of 3,4-epoxy-paracetamol as the toxic metabolite

(Andrews et al.,1976) was negated by the investigations of

Hinson et al. (1980) and Hinson and Gillette (1980). They

demonstrated through mass spectral studies that atmospheric

oxygen was not incorporated into the paracetamol-glutathione

adduct, which would otherwise be expected were paracetamol to

form an arene oxide in the 3,4-position followed by

rearrangement to NAQ prior to conjugation (Hinson et al.,1977).

Also when p-18-0-paracetamol was used as a substrate, all of the

18-0 was retained in the paracetamol-glutathione complex (Hinson

et al.,1979c). The evidence does not indicate 3,4-epoxidation

of paracetamol, but similar experiments with phenacetin did

reveal 3,4-epoxidation as one mechanism of microsomal activation

of phenacetin to a reactive metabolite (Hinson et al.,1979a).

A theory of free radical mediated toxicity has been proposed but

is yet to be proved (Andrews et al.,1976).

1.3.1.2.3. N-ACETYL-p-BENZOQUINONEIMINE

The other proposed toxic intermediate for paracetamol in the

literature to date is the electrophile N-acetyl-p-

benzoquinoneimine (NAQ). NAQ, which covalently binds with

protein (Mulder et al.,1978) and glutathione (Hinson et al.,

1979c), has been generally accepted as the most likely ultimate

toxic intermediate of paracetamol (Hinson et al., ,1979a; Nelson

et al.,1980; Calder et al.,1981). Though it was supposedly

formed from NHAPAP, a toxic reactive species in itself (Jollow

et al.,1974a), the evidence (Hinson et al.,1979a; Nelson et al.,

1980) indicates that it must be formed from still another

reactive source because NHAPAP, with a relatively slow

decomposition (Hinson et al.,1979a) which would enable it to be

detected if it were formed, is not an intermediate involved in

the metabolism of paracetamol in vitro (Hinson et al.,1979a;

Nelson et al.,1980) or in vivo (Gemborys and Mudge,1981).

Calder et al. (1981) further confirmed that no N-hydroxylated

metabolites resulted from paracetamol administration and

postulated the concept of hepatotoxicity and nephrotoxicity

being directly mediated by an oxidation of paracetamol to the

toxic reactive intermediate NAQ by the cytochrome P-450-

dependent mixed function oxidase. However, NHAPAP could still

be a reactive toxic metabolite of phenacetin, as it has been

found to be present as a metabolite of phenacetin and though

believed earlier to be formed by the N-hydroxylation of

paracetamol, it has since been shown to be formed by the

subsequent de-ethylation of N-hydroxylated phenacetin (Hinson et

al.,1979a). An in vivo measurement of NHP formation would

therefore reflect the extent to which postulated toxic

metabolites of paracetamol could be formed from phenacetin.

1.3.1.3. p-AMINOPHENOL

p-Aminophenol (PAP), identified as a urinary . metabolite of

paracetamol and NHAPAP in the hamster (Gemborys and Mudge,1981)

and known to be highly nephrotoxic though relatively non-

hepatotoxic (Green et al.,1969; Calder et al.,1971; Crowe et

al.,1979; Newton et al.,1982) could be of toxicological interest

as well. It has of late been considered to be an immediately

reactive nephrotoxic compound (Calder et al.,1979).

1.3.1.4. CONJUGATES OF PARACETAMOL

The major urinary metabolites of paracetamol, paracetamol-

glucuronide, paracetamol-sulfate and 3-mercapturate-paracetamol,

occur in all species (Gemborys and Mudge,1981), with other

metabolites being identified uniquely to a particular species.

These are 4-hydroxyglycoanilide in rats (Smith and Griffiths,

1976), 3-cysteinyl-paracetamol in man and mice (Mrochek et al.,

1974; Whitehouse et al.,1977), 3-sulfoxymethyl-paracetamol in

hamsters (Wong et al.,1976), 3-thiomethyl-paracetamol in man and

dog (Klutch et al.,1978), 3-methoxyparacetamol in man (Andrews

et al.,1976) and 3-hydroxyparacetamol in man (Andrews et al.,

1976; Mrochek et al.,1974).

1.3.2. p-PHENETIDINE

N-deacetylation is considered to be an essential step in the

precipitation of haemotoxicity by acetanilide analogues

(Mitchell et al.,1973c). The N-deacetylation of phenacetin

gives p-phenetidine (PN) as shown in Fig. 1. This is the second

major direct metabolite of phenacetin. The subsequent

metabolism of PN has not been adequately reported, although it

has been suggested that it undergoes oxidation to quinones and

aromatic nitrosoamines and it is known to cause

methemoglobinemia by the formation of haemotoxic metabolites

NHPN, 2HPN and PAP (Kiese,1966; Uehleke,1973). Evidence of its

existence as the second major metabolite of phenacetin and its

disappearance in the metabolic process has been recorded using

isolated hepatocyte systems (McLean,1978). The de-ethylated, N-

hydroxylated, nitrosated and 2-hydroxylated metabolites of PN

have been reported (Brodie and Axelrod 1 1949), but their

contribution to nephrotoxicity has not been determined.

1.3.3. 2-HYDROXYPHENACETIN, 3-HYDROXYPHENACETIN

and 2-HYDROXYPHENETIDINE

The hydroxylated products of phenacetin, 2-hydroxyphenacetin

(2HP), 3-hydroxyphenacetin (3HP) and 2-hydroxyphenetidine (2HPN)

have been reported (Buch et al.,1966; 1967; Klutch et al.,1966)

earlier. 2HP was devoid of antipyretic activity in rats and did

not cause methemoglobinemia in dogs and in rats no abnormal

gross or histological changes in kidney function or structure

were produced on oral administration for prolonged period

(Burns and Conney,1965). No toxicity has been reported with 2HP

in the concentrations at which it is usually present. However,

isolated cases of toxicity have been reported for 2HPN. Shahidi

and Hemaidan (1969) presented a case where large amounts of 2HPN

were present in the urine of a female patient who developed a

severe hemolytic reaction. These abnormalities in respon-se were

rare and familial.

1.3.4. N-HYDROXY METABOLITES

Many toxic substances are generally detoxified within biological

systems by conjugation with glucuronic acid by UDP-

glucuronyltransferase, or sulfation by sulfotransferase (Mulder

and Scholtens,1977), or combination with glutaihione (GSH)

(Mitchell et al.,1973b; 1974; Jollow et al.,1974b). Whether a

compound would eventually prove to be toxic, would therefore

depend on the extent to which the conjugating enzymes were

active and the quantity of glutathione available for

electrophilic "mopping up".

In recent years attention has been focussed on the toxicity

caused by arylamines. The arylamines have earned notoriety as

compounds capable of undergoing hydroxylation by hepatic mixed

function oxidases to yield the proximal carcinogenic N-

arylhydroxylamines and N-arylhydroxamic acids (Baldwin and

Smith,1965; Miller and Miller,1966a; Kiese,1966; Miller,1970;

Weisburger and Weisburger,1973; Miller,1978).

Several compounds possessing an N-hydroxyl group in their

molecules, have been reported as carcinogenic compounds. N-

arylhydroxylamines such as N-hydroxy-2-naphthylamine, N-hydroxy-

4-aminobiphenyl (Radomski and Bri11,1970; 1971; Radomski et -

al.,1977) and N-arylhydroxamic acids such as N-hydroxy-2-

acetylaminofluorine (Miller et al.,1961; Razzouk et al.,1977),

whose activated esters such as the sulfate, acetate and

glucuronide, are strong electrophiles. These compounds have been

shown to possess carcinogenic character reflected in their

ability to bind to the nucleophilic groups in cellular

macromolecules (Scribner and Naimy,1973; Miller et al.,1974;

Miller and Miller,1976).

In specific cases of N-arylhydroxylamines and N-arylhydroxamic

acids such as N-hydroxy-2-acetylaminofluorene (DeBaun et al.,

1970), N-hydroxy-N-methyl-4-aminobenzene (Kadlubar et al.,1976),

N-hydroxy-4-acetylaminobiphenyl (Kriek,1971), N-hydroxy-N,N-

diacetylbenzidine (Morton et al.,1980), N-hydroxy-2-

acetylaminophenanthrene (Scribner and Naimy,1973) and N-

hydroxyphenacetin (Mulder et al.,1977), sulfation increased the

toxicity of the parent compounds.

The sulfate conjugates produced were highly reactive with tissue

nucleophiles. Contrary to expectations of the detoxification

role of sulfation in biological processes, sulfation of certain

N-arylhydroxylamines and N-arylhydroxamic acids results in the

formation of their reactive entities.

The esterification of N-arylhydroxylamines and N-arylhydroxamic

acids has been therefore regarded as an activation pathway

leading to carcinogenesis for N-hydroxy compounds (Weisburger,

1978). However, the carcinogenic character of a compound is not

entirely explained by the occurrence of an N-hydroxyl group

within its molecular structure (Weisburger,1978).

Phenylhydroxylamine and N-hydroxysuccinimide are examples of

such non-carcinogenic compounds.

1.3.4.1. N-HYDROXYPHENACETIN : A PROXIMAL CARCINOGEN

NHP is an important metabolite of phenacetin, in terms of its

carcinogenicity. It has been suggested to be the metabolite of

phenacetin most likely to induce tumors due to its chemical

similarity to the known carcinogenic N-arylhydroxamic acids

(Calder and Williams,1975). It has also been suggested as the

'metabolic product of phenacetin which could best account for the

covalent binding of phenacetin to cell protein (Nery,1971a) and

as an intermediate in the formation of other products of

phenacetin metabolism (Nery,1971b; Calder et al.,1974). The

mechanism of the renal and hepatic toxicity of phenacetin has

been proposed to occur in two steps, N-hydroxylation followed by

conjugation and subsequent decomposition to a reactive

intermediate (Mulder et al.,1977; 1978). The same metabolic

sequence activates the carcinogen, 2-acetylaminofluorene.

Evidence of in vitro N-hydroxylation by liver microsomes has

been demonstrated in hamsters (Hinson and Mitche11,1976),

rabbits (Fischbach et al.,1977), mice (Kapetanovic et al.,1979)

and in vivo experiments in rats (McLean et al.,1981). Evidence

of conjugation with sulfate and glucuronide in vitro by rat

liver enzymes, and subsequent decomposition has been contributed

by Mulder et al. (1977,1978). Calder et al. (1976) showed that,

. in chronically treated rats NHP induced neoplasia and tumors of

the liver were a direct manifestation of its carinogenicity. A

quantitative estimation of NHP over a prolonged study period

would therefore provide information on the increased formation

or accumulation of this metabolite, which may ultimately relate

to the toxicity of the parent analgesic compound, phenacetin.

1.4. CHRONIC DOSING STUDIES

Since toxicity from phenacetin is only seen after prolonged

administration of the drug, progressive monitoring of metabolic

changes over extended periods, accompanied by pathophysiological

surveillance and toxicokinetic evaluation would prove helpful in

determining the cause of phenacetin-induced carcinogenicity and

nephrotoxicity.

The effect of the size of the chronic dose on metabolism and or

on the development of carcinoma or nephropathy could also be

included in such investigations. The development of tolerance

or increased susceptibility to phenacetin-toxicity could further

be closely examined as there have been reports of related

analogues of phenacetin being able to protect animals from

paracetamol-induced hepatotoxicity (Kapetanovic,1979). Chronic

dosing with phenacetin itself has been shown to cause tolerance

and protection from hepatotoxicity (Boyd and Hottenroth,1968;

Boyd,1971; Carro-Ciampi,1971; 1972; Kapetanovic and Mieyal,

1979). The metabolic interference likely to be caused by other

drugs or compounds co-administered with phenacetin could also be

investigated.

1.4.1. ALTERED METABOLISM WITH CHRONIC DOSING

Carro-Ciampi (1972), in chronic studies with phenacetin, further

demonstrated tolerance to phenacetin-induced hypothermia in both

albino rats and guinea pigs, after repeated daily

administration. Irrespective of the daily dose used, tolerance

developed in guinea pigs more slowly than in albino rats. In

acute phenacetin toxicity death is due to hypothermic coma as

well as cyanosis, respiratory depression and cardiac arrest and

therefore, when tolerance develops to phenacetin hypothermia,

animals are able to survive normally lethal doses of the drug

(Boyd,1959; 1960; Boyd et al., 1969; Carro-Ciompi, 1971).

These chronic-dose studies (Carro-Ciampi, 1972) indicated a

halving of plasma levels of phenacetin within 10 days of

initiation of phenacetin administration. This effect developed

more slowly in guinea pigs, and was accompanied by a marked

increase in PN levels. This would account for the chronic

hemolytic anemia (Schnitzer and Smith, 1966) and

methemoglobinemia (Brodie and Axelrod,1949) seen more commonly

in guinea pigs.

One hundred day LD 50 studies provide further examples of how

chronic dosing studies (Boyd and Hottenroth,1968; Boyd and

Carro-Ciampi,1970; Boyd,1971) undertaken to stress the

importance of toxicity caused by prolonged administration of

phenacetin, could be beneficial.

1.4.2. INFLUENCE OF DOSE SIZE, ADMINISTRATION ROUTE,

SPECIES VARIATION AND DURATION OF TREATMENT

Few investigations have been carried out to examine factors such

as dose, administration route and species which could probably

affect the metabolism of phenacetin and its alleged toxicity

with chronic use. Smith and Timbrell (1974) carried out such

investigations and found the drug to be largely metabolized in

the rat, rabbit, guinea pig, ferret and man by oxidative

deethylation and deacetylation. Minor pathways of aromatic

hydroxylation and cysteine conjugation were also present.

In species-related metabolism of phenacetin (Smith and Timbrell,

1974), deacetylation was highest in the rat (21% of dose),

aromatic hydroxylation to 2-hydroxyphenacetin was highest in the

ferret (6% of dose) and formation of the 3-cysteine conjugate

was highest in the rabbit (8% of dose). The pattern of

conjugation was such that glucuronidation was predominant in the

rabbit, guinea pig and ferret while sulfate conjugation was the

major route of metabolism in the rat. The metabolic profile

differed between large and small oral doses but showed no

appreciable differences whether the drug was given orally or

intraperitoneally.

Different species of animals are affected by methemoglobinemia

caused by phenacetin to varying degrees (Lester, 1943; Welch et

al., 1966). This is probably because several deicetylated

derivatives of phenacetin, (NHPN, 2HPN and PAP), which are known

to oxidize hemoglobin (Uehleke, 1973), are formed to varying

degrees in the different species (Smith and Timbrell, 1974).

In studies of Smith and Griffiths (1976) metabolism of 14-C-

phenacetin in rats fed the drug in their diet over a 3 month

period, was examined and compared with that in a control group

of rats receiving only a single dose of the drug. The major

metabolite was APAP-SULF in both groups of animals. Variance

was noted in glucuronidation between the groups. Other

metabolites seen were APAP, p-hydroxyglycoanilide, p-

ethoxyglycoanilide and 2HP. Excretion of total radioactivity

was proportionally reduced when larger doses of phenacetin were

given.

1.4.3. INFLUENCE OF CO-ADMINISTERED DRUGS

More recently chronic dosing studies with phenacetin, caffeine

and aspirin singly or in combination were undertaken by Macklin

and Szot (1980) in mice. Histopathological changes indicating

mild progressive renal papillary necrosis occurred in the

urinary tract with earliest changes observed in those animals on

the highest dose of phenacetin. Sulfhemoglobinemia was induced

in all animals subjected to treatment with phenacetin alone or

in combination with the other agents. Failure to demonstrate

carcinogenicity even at these toxic doses of the drugs was in

agreement with the negative results obtained in similar studies

in mice described in the NCI Technical report (No.67,1978) cited

by Macklin and Szot (1980), rats (Woodard et al., 1965; Schmahl

and Reiter 1954, NCI Tech. report,No.67,1978) and dogs (Schmahl

and Reiter,1954; Woodard et al., 1965). Yet, other chronic

dosing studies with phenacetin in rats did present evidence of

carcinogenicity (Johannson and Angervall, 1976; 1979; Isaka et

al., 1979).

The disagreement in these results of very similar experiments

was suggested to be due to a formulation shortcoming (Macklin

and Szot, 1980). Administration of the dose through the diet in

the studies which produced tumours involved pelletization of the

drug along with the other food ingredients. This process

requires high temperatures of about 80 C, during which the

formation of N-oxidation reactive products was claimed to occur.

Nitrosocompounds have been known to induce tumours, particularly

of the nasal cavities (Magee et al., 1976) as well as the

urinary tract, urinary bladder, renal and mammary glands (Magee

and Barnes,1967; Magee et al.,1976; IARC, 1978; Ito et al.,

1971).

Johannsson (1981) studied long term treatment with phenacetin,

phenazone and caffeine, individually and in combination. Renal

pelvic tumours occurred only in rats treated with phenacetin, or

phenazone alone or in combination with caffeine. Half of the

rats treated with phenacetin, phenazone and caffeine in

combination developed hepatomas which were considered to be a

result of the altered metabolism of phenacetin caused by

phenazone and caffeine. It was postulated that this altered

metabolism ultimately increased the production of N-

hydroxyphenacetin, a known liver carcinogen (Johansson,1981).

1.4.4. INFLUENCE OF AGE AND SEX

Factors of age- and sex-related differences within a species are

also likely to affect the metabolism of phenacetin. The

hepatotoxicity of paracetamol has been investigated with

reference to age of the experimental mice by Hart and Timbrell

(1979) who found paracetamol to be less toxic in neonatal mice

than in adult animals. This suggests that the development of

the ability to detoxify reactive metabolites precedes the

development of the enzyme systems producing them, because

glutathione levels have been shown to be higher than the levels

of P-450 in neonates. Green and Fischer (1981) established from

similarly oriented research that age-related changes in

paracetamol metabolism in rats, especially in the extent of

glucuronidation or sulfation, are complex and depend on the dose

of the drug and sex of the animal. The influence of age and sex

on phenacetin metabolism have not been investigated in the

present study.

1.4.5. INFLUENCE OF SULFATION INHIBITION

Interference with metabolism by the administration of compounds

capable of inhibiting metabolic pathways in order to study the

ensuing metabolic changes has been carried out previously

(Meerman et al., 1980; Meermen and Mulder 1981; Mulder and

Scholtens, 1977). The sulfation pathway, which is a predominant

metabolic pathway for phenacetin in the rat, has been

selectively

inhibited by 2,6-dichloro-4-nitrophenol,

salicylamide and pentachlorophenol with a simultaneous increase

in glucuronidation of the substrate, harmol (Mulder and

Scholtens, 1977). It has been previously postulated that N-0-

sulfation, following N-hydroxylation, has been responsible for

the production of the reactive metabolites of phenacetin which

combine with tissue macromolecules (Mulder et al.,1977; 1978).

The analogous N-0-sulfation product of N-hydroxy-2-

acetylaminofluorene has been similarly implicated in the

precipitation of toxicity (DeBaun et al.,1970). Inhibition of

sulfation would therefore be expected to obviate toxicity

claimed to be caused by the N-0-sulfate conjugate of NHP. This,

therefore, warrants investigation with a further study of any

accompanying metabolic changes that occur when sulfation is

inhibited.

NHCOCH3 NHCOCH3

0C2145 _ _ P 3.4€

NHCOCH3

-

OH NCOCH3

0C2Hs

NHP

N COC H 3

-o N AO

NHCOCH3

NHCOCH3

HO

_

GSH

OH

APAP—GSH

OH

( SULF) GLUC-0-NCOCH3

GSH

OH

N•0-GLUC: N-O-SULF

OH NCOCH3

2.

OH

NHAPAP

GSH

FIG. 2: Postulated mechanisms of phenacetin-induced toxicity

1.5. POSTULATED MECHANISMS OF PHENACET1N-INDUCED TOXICITY

Although phenacetin is referred to as a toxic drug, the precise

mechanism of its toxicity is still to be determined. Studies in

hamsters (Mitchell et al.,1975) have postulated conversion to

chemically reactive metabolites via several routes of metabolism

(Hinson et al.,1979b; 1979c; Nelson et al.,1981). At least

three different methods of activation have been suggested by

which phenacetin is expected to be converted to chemically

reactive metabolites responsible for its toxicity (Fig.2).

i) Phenacetin undergoes oxidative deethylation by hepatic

enzymes to form paracetamol, which in turn is converted to a

chemically reactive nephrotoxic and hepatotoxic metabolite,

probably NAQ (Calder et al.,1981).

ii) Hepatic enzymes convert phenacetin to an arylating

metabolite : plienacetin-3,4-epoxide (Hinson et al.,1977).

iii) Hepatic enzymes convert phenacetin to N-hydroxyphenacetin

(Hinson and Mitche11,1976), which could be conjugated as the N-

0-sulfate and N-0-glucuronide, both reactive electophiles

(Mulder et al.,1977,1978). Additionally or alternatively NHP

could be converted to NAQ (Calder et al.,1974).

In the light of recent research, NHP has been considered to be

the metabolite of phenacetin most likely to induce nephropathy

and carcinogenesis (Calder et al.,1973; 1976; Calder and

Williams,1975; Nery,1971c). The urinary excretion of NHP could

therefore be indicative of the probable carcinogenicity and

nephrotoxicity of phenacetin.

1.6. SCOPE OF THE PRESENT IN VIVO CHRONIC DOSING STUDY

The present chronic study was therefore undertaken to elucidate

more clearly the metabolism of phenacetin and any changes in

metabolism after prolonged administration of the drug. Such

factors could be indicative of the mechanism of phenacetin-

induced toxicity.

An in vivo chronic study in rats was undertaken to monitor the

excretion levels of the various metabolites of phenacetin,

during a period of prolonged administration of the drug, at

regular (weekly) intervals.

NHP has been implicated as a likely proximal carcinogen of

phenacetin. Levels of this probably carcinogenic, reactive

intermediate, which were not determined in other chronic studies

(Smith and Griffiths, 1976; Smith and Timbrell, 1974), were the

focus of the present study.

The effect of the size of dose has already been investigated

with reference to the extent of metabolism of the drug (Smith

and Timbrell, 1974) and its toxicity (Boyd and Hottenroth, 1968;

Boyd, 1971), but these studies had not measured the significant

metabolite, NHP. The present study also sought to examine the

influence of size of dose on the formation of NHP.

The influence of aspirin and caffeine on the metabolism of

phenacetin was examined. It was of particular interest to

detect any alteration in metabolism of phenacetin when co-

administered with these drugs.

The effect of chemicals likely to interfere with certain major

pathways of phenacetin metabolism (such as sulfation) was of

interest in this study. The intention was to infer the extent

of saturation of the existing alternative pathways by

suppressing the sulfation pathway.The possibility of direct

interference with the metabolism of phenacetin by these

chemicals was also of interest. An inhibition of sulfation

would also be expected to result in diminished formation of the

N-0-sulfate conjugate of NHP, a known reactive metabolite.

1.7. ELUCIDATION OF THE METABOLISM OF p-PHENETIDINE IN VITRO

The toxicity caused by PN, which is known to be hemolytic, could

be direct or through a subsequent metabolite.

Although in vitro systems using microsomes have been used to

study the metabolism and toxicity of several compounds,

including phenacetin and paracetamol (Hinson et al., 1979a;

Nelson et al., 1980), metabolic studies using PN as a substrate

(Buch et al., 1967) which indicated its conversion to 2-HPN, did

not reveal details of its further metabolism. A study involving

an in vitro examination of PN metabolism was therefore also

undertaken in the present work.

1.8. SELECTION OF AN IN VITRO SYSTEM

Berry and Friend (1969) successfully developed enzymatic

techniques for the isolation of viable hepatocytes and thereby

introduced a reliable in vitro system for studying the

metabolism or toxicity of xenobiotics.

Several substrates have been studied using isolated hepatocytes.

Benzpyrene (Cantrell and Bresnick, 1972), alprenolol (Moldeus et

al., 1974), biphenyl (Wiebkin et al., 1976), ethyl morphine

(Erickson and Holtzman, 1976), amylopyrene, dansylamide, quinine

(Hayes and Brendel, 1976), barbiturates (Yih and van Rossum,

1977), amphetamine (Hirata et al., 1977) and methotrexate (Horne

et al., 1976) are a few examples.

Isolated hepatocytes were chosen as the in vitro technique for

the present study because this system has already proven its

suitability for studies of drug metabolism and toxicity (Thor et

al.,1978a; 1978b; Moldeus,1978). This is because more cellular

properties are retained by isolated hepatocytes in comparison

with microsomal preparations. Both cytochrome P450-dependent

oxidation reactions (Moldeus et al.,1974; Yih and van Rossum,

1977) and subsequent conjugation reactions (Wiebkin et al.,1976;

Billings et al., 1977) are possible. Most importantly, better

correlation with in vivo results of drug metabolism has been

established (Billings et al., 1977; Yih and van Rossum, 1977)

by using isolated hepatocytes in comparison with 900xg

supernatant.

1.9. AIMS OF THE PRESENT STUDY

i) Study the metabolism of chronically administered phenacetin

in the rat.

ii) Examine the influence of dosage size on metabolism.

iii) Investigate the effect of sulfation inhibition on

phenacetin metabolism and comparatively assess the metabolic

alterations, if any, seen on administration of phenacetin

acutely and chronically.

iv) Detect and determine any alteration in phenacetin

metabolism when phenacetin and aspirin are chronically co-

administered.

v) Detect and determine any alteration

in .phenacetin

metabolism when phenacetin and caffeine are chronically co-

administered.

vi) Follow the further metabolism of deacetylated phenacetin

(p-phenetidine) in an in vitro system and to account for its

disappearance.

CHAPTER 2. EXPERIMENTAL

2.1. MATERIALS

p-Phenetidine (Hopkin & Williams Ltd, England) was redistilled,

b.p. 251 C. p-Nitrosophenetole was synthesized (Sec. 2.1.1.).

N-Hydroxyphenacetin and deuterated N-hydroxyphenacetin were

gifts from Dr. S. McLean (School of Pharmacy, University of

Tasmania). 2-Hydroxyphenetidine, N-acetyl-4-aminobenzoic acid

and N-butyry1-4-aminobenzoic acid were gifts of Mr. M. Veronese

(School of Pharmacy, University of Tasmania).

The glucuronide, sulfate, cysteinyl and mercapturate conjugates

of paracetamol were gifts from Dr. K. Henderson (Sterling

Winthrop, Newcastle-upon-Tyne, Great Britain).

Extract of Helix pomatia (beta-glucuronidase plus arylsulfatase)

was obtained from Boehringer, Mannheim, Germany. Fluorescent

silica gel (Schleicher and Schull type G7, size 10-40 ) was

coated (0.25 mm thick) on to glass thin layer chromatography

plates (20 x 20cm).

Collagenase Type IV and Bovine Serum Albumin (BSA) essentially

fatty acid free (Sigma Chemical Company, U.S.A.) were employed

in hepatocyte isolation.

Diazomethane was prepared fresh when required for methylation

from p-tosylsulfonylmethylnitrosamide by the method of Vogel

(1956), and used as the ethereal solution.

Drugs were of B.P. grade. All other chemicals and solvents were

of A.R. grade and purchased commercially.

2.1.1. SYNTHESIS OF p-NITROSOPHENETOLE

p-Nitrosophenetole was synthesized by a modification of the

method of Vogel (1959). p-Nitrophenetole (500mg, 0.003 mole)

was dissolved in ethanol (99.5% v/v, 16m1). Ammonium chloride

(640 mg, 0.012 mole) dissolved in water (3m1) and zinc powder

(780mg, 0.012 mole) was added.

The reaction mixture was stirred vigorously at room temperature

for 90 min and checked for completion by thin layer

chromatography on a silica gel coated miniplate developed in

ether and visualized with ferric chloride (2.5% w/v in M/2 HC1).

The reaction mixture was filtered and the precipitate washed

with ice cold ethanol. The filtrate was transferred to a

separating funnel. A saturated solution of sodium chloride was

added and the mixture shaken thoroughly.

Chloroform (2x25 ml) was used to extract the salted-out p-

nitrosophenetole and the pooled chloroform extract washed with

ice cold water (2x25 ml). The chloroform extract was dried over

anhydrous magnesium sulfate for 1 hour with frequent agitation,

and evaporated off under vacuum in a rotary evaporator. This

left a residue of p-nitrosophenetole which was later

recrystallized from benzene and characterized by mass

spectrometry. The recrystallized p-nitrosophenetole was pure.

It produced a single spot when thin layer chromatographed

(silica gel-ether) and one peak when gas chromatographed under

conditions described in Fig. 6.

2.2. ANIMALS

A Hooded Wistar strain of rats fed on a standard laboratory diet

with water ad libitum were used in all present studies. The

rats weighed approximately 200 g and were 4 weeks old when used.

2.3. IN VITRO STUDY

An in vitro system using freshly isolated hepatocytes from the

rat was employed.

2.3.1. HEPATOCYTE ISOLATION

A modified method incorporating the techniques of Berry & Friend

(1965) and Seglen (1972; 1973a; 1973b) was adopted (Fig. 3).

The rat was anaesthetised with pentobarbitone (65 mg/kg;

prior to surgery. After regular respiration was established, a

mid-ventral incision was made to expose the liver and heparin

(0.1 ml of 25,000 units/ml) was injected into the inferior vena

cava to prevent blood coagulation.

The hepatic portal vein was cannulated with a tube delivering a

flow of 5 ml/min of calcium-free Krebs-Henseleit buffer

(glucose 10mM, carbogen equilibrated, pH7.4, 37 C).

Immediately after cannulation the buffer flow was increased to

30 ml/min, the liver was flushed of all blood and the superior

vena cava was cut open to allow for the drainage of the buffer,

in situ.

INTERCELLULAR DISRUPTION

INCUBATION

WITH SUBSTRATE

DECAPSULATION, DISPERSION

SAMPLING

HARVESTING

CENTRIFUGATION

RECOVERY

SUPERNATANT I viability count

RECONSTITUTION OF

ISOLATED HEPATOCYTES ANALYSIS

ANAESTHETIZATION

NEPA R IN I Z AT ION

CAN NULA TION

MOUNTING

PERFUSION

pH 7-4, 37°C

carbogen

Ls Ca-free Krebs - Henseleit, 15 min

Ls Krebs-Henseleit, 5 min

Krebs-Henseleit collagenase, 45 min

FIG. 3: Scheme for the isolation and use of hepatocytes

The liver was excised from its connective tissue and mounted in

a perforated plastic receptacle. After 12 min the buffer was

replaced with Krebs-Henseleit buffer (glucose 10mM, carbogen

equilibrated, pH 7.4, 37 C).

After 5 min, collagenase (25 mg in 5 ml buffer) was added to

give a final concentration of 0.05 % in the perfusate (50 ml),

which was circulated for a further 45 min. The liver showed

visible signs of disruption after this period.

The liver was subsequently transferred into Krebs-Henseleit

buffer containing BSA (0.1%w/v). The capsule was removed and

hepatocytes gently shaken free from the connective tissue.

The suspension of cells and tissue was sieved through nylon

gauze which retained the connective tissue. The cell suspension

was allowed to stand for a few minutes and the sedimented cells

were harvested by decanting off the supernatant liquid.

After reconstituting with BSA-Krebs Henseleit buffer, the cells

were allowed to recover under carbogen, in an orbital shaker

(37 C, 30 min, 120 osc/min).

Trypan blue (0.2%) was used to estimate the viability count

which was done in a Neubauer chamber. Viability was between 68%

and 75%. Cell yield was between 1.3 x 108 to 1.6 x 108.

2.3.2. INCUBATION AND SAMPLE COLLECTION

The isolated hepatocytes from healthy untreated rats were

reconstituted to give a viable cell concentration of 2.5 x

10 6 /m1 in Krebs-Henseleit buffer (BSA 1% , pH 7.4). An aliquot

(5m1) was collected to serve as the blank sample. The substrate

of •p-phenetidine (3.4 mg in 100 ul ethanol/water:30/70) was

added to the cell suspension (50 ml) in an incubation flask,

giving a PN concentration of 0.5 mM, found suitable for

metabolic studies by McLean (1978).

A zero time sample aliquot (5 ml) was removed, carbogen flushed

through the incubation flask and the flask replaced in the

orbital

shaker bath (180 osc/min, 37 C), until the next

collection was due. Samples were collected as scheduled in

Table 1, and the carbogen replenished in the flask after each

sample removal.

The aliquots were collected in 15 ml centrifuge tubes kept in

ice. The aliquot suspensions were centifuged immediately (2500

rpm for 10 min) and 2 ml duplicate samples of supernatant were

transferred to centrifuge tubes, frozen immediately in liquid

nitrogen and stored for assay at -20 C.

The samples were analysed as outlined in the analytical scheme

for metabolites of p-phenetidine (Sec. 2.4. and Fig. 4) and the

results of the analyses are presented in Table 1.

OXIDATION

K3Fe(CN)6

EXTRACTION

CCl4

SUPERNATANT

from Incubate

+ internal + standard

internal + standard

ENZYMATIC HYDROLYSIS

fi-glucuronidase + arylsulfatase

37°C 16hr

BUTYRYLATION

NaHCO3,

butyric anhydride

1. EXTRACTION

CH2Cl2

GC

APAP PAP PN

ACID HYDROLYSIS

10N HCl, 100°C, 1hr

PHENOXAZONE FORMATION

pH 8 -9, room temp.

EXTRACTION

CH Cl3

COLOR DEVELOPMENT

1N HCl, 70°C

SPECTROPHOTOME TRY

X 580 nm

2HPN

I GC I NOPN NHPN

FIG. 4: Analytical scheme for the in vitro metabolites of p—phenetidine

2.4. ANALYSIS OF THE METABOLITES OF p-PHENETIDINE

Analytical methods for the extraction and analysis of the

metabolites of p-phenetidine present in the supernatant of the

incubate were developed.

2.4.1. DETECTION AND MEASUREMENT OF PHENACETIN, PARACETAMOL,

p-AMINOPHENOL AND p-PHENETIDINE

2.4.1.1. HYDROLYSED SAMPLES

An aliquot (2 ml) of incubate supernatant was buffered to a pH

of 5.2 with 200 ul acetate buffer (1.1M, pH 5.2), in a 15 ml

centrifuge tube. Helix pomatia extract (100 ul) and the

internal standard, p-toluidine (TDN, 50 ug in 50 ul methanol),

were added. After vortexing briefly (10 sec) the sample was

hydrolysed (37 C, 16 hr).

Sodium bicarbonate (120 mg) was added to the sample after

hydrolysis and vortexed until dissolved to obtain a neutral pH.

Butyric anhydride (25 ul) was added and the sample vortexed

frequently over a period of 1 hour after which butyrylation was

complete.

The sample was then partitioned with dichloromethane (3 ml),

vortexed (30 sec) and centrifuged (2500 rpm for 15 min). The

aqueous upper layer was discarded and the dichloromethane

extract decanted off after freezing with liquid nitrogen. The

dichloromethane extract was further concentrated to 50 ul under

a gentle stream of nitrogen at room temperature. Finally, 1 ul

of the concentrated extract was gas chromatographed using

conditions shown in Fig. 5. Detection of the metabolites was by

comparison with authentic standards. The concentrations of the

metabolites were determined by reference to the standard curves

for phenacetin (linearity: 0.5 ug/ml - 10 ug/ml), paracetamol

(linearity: 0.5 ug/ml - 10 ug/ml), p-aminophenol (linearity: 0.5

ug/ml - 5 ug/ml) and p-phenetidine (linearity: 5 ug/ml -

15Oug/m1), using peak height ratios to internal standard.

2.4.1.2. UNHYDROLYSED SAMPLES

The enzyme incubation step was omitted. p-Toluidine was added

and samples were butyrylated and extracted for analysis as

above.

2.4.2. DETECTION AND MEASUREMENT OF p-NITROSOPHENETOLE

AND N-HYDROXYPHENETIDINE.

An aliquot of incubate supernatant (2 ml), was transferred into

a 15 ml centrifuge tube. p-Bromoaniline (BA, 50 ug in 50 ul

methanol), the internal standard, was added. The method of

Kiese and Renner (1963) using potassium ferricyanide to oxidize

the N-hydroxyphene-tidine to p-nitrosophenetole was employed.

Potassium ferricyanide (25 ug in 25 ul water) was added and the

sample vortexed for 10 sec. Carbon tetrachloride (1 ml) was

vortexed for 60 sec with the sample, to extract the required

metabolites. The sample mixture was then centrifuged (2500 rpm

for 15 min) and the upper aqueous layer discarded. The sample

was frozen and the organic phase decanted and concentrated under

a stream of nitrogen. Finally 1 ul of the extract was gas

chromtographed using conditions shown in Fig. 6. Detection was

by comparison with the authentic standard and concentrations

could be determined by reference to the standard curve for p-

nitrosophenetole (linearity: 0.5 ug/ml - 10 ug/ml) using peak

height ratios to internal standard.

2.4.3. DETECTION AND MEASUREMENT OF 2-HYDROXYPHENETIDINE

A method based on that of Shahidi and Hemaidan (1969) was used

to measure 2-hydroxyphenetidine. An aliquot of incubate

supernatant (4 ml) in a 30 ml stoppered centrifuge tube, was

acid hydrolysed (1 ml 10 M HC1, 100 C, 1 hr). The hydrolysed

sample was neutralized with 5M sodium hydroxide (2 ml) and the

pH adjusted to 8-9 with sodium bicarbonate. After the sample

was vortexed for 30 sec it was kept at room temperature for 3 hr

to allow for the formation of the phenoxazone. The tubes were

cooled in ice and extracted sequentially with chloroform

(2x20 ml). The chloroform extracts were pooled and evaporated

to dryness under vacuum in a rotary evaporator. Color

development was effected through the addition of preheated

(70 C) 1M hydrochloric acid (2 ml). The absorbance was read at

580 nm on a Bausch and Lomb Spectronic 20 spectrophotometer.

Concentrations were determined by reference to a standard curve

for 2-hydroxyphenetidine (linearity: 0.5 ug/ml - 25 ug/ml).

a.

a.

a. 4 a. 4

BLANK STANDARDS SAMPLE

a.

DE

TE

CTO

R R

ES

PON

SE

a. 4 a.

32, .32

.258 .32 .258 .258

.32 1 ..16 .512 .512

0369 12 636 9 12 Oa 9 12

TIME ( mm)

FIG. 5: GC trace for the metabolites of p-phenetidine in isolated hepatocytes

Metabolites were derivatized (Section 2.4.1.).

Hewlett Packard gas chromatograph 5700A;

Column : 0V210 (3% on 100/120 diatomite MQ, glass, 90cm x 2mm ID);

col : 200°C; inj : 250°C; det. : 250oC;

Carrier gas : N 2 (30 ml/min)

FID gases : Air (240 ml/min), 112 (30 ml/min).

BLANK STANDARDS SAMPLE

DE

TEC

TOR

RES

PON

SE

O 2 4 6

a. 0

a.

0 2 4 6

TIME ( m n )

•■•■••

O 2 4 6

FIG. 6: GC trace for the metabolites of p-phenetidine in isolated hepatocytes

Hewlett Packard gas chromatograph 5700A:

Column : OV-225 (3% on 100/120 diatomite MQ, GLT, 1.0m x 1.8mm ID);

col. : 132°C; inj. : 150 °C; det. : 200 °C;

Carrier gas : N2 (20 ml/min);

FID gases : Air (240 ml/min), H 2 (30 ml/min).

2.5. IN VIVO STUDY

2.5.1.1. CHOICE OF DOSE

Chronic oral daily doses of phenacetin (50 mg/kg), aspirin (50

mg/kg) and caffeine (10 mg/kg) were chosen on the basis of

relevance to present day human consumption of the drugs. The

dose of 100 umol/kg pentachlorophenol, a known inhibitor of

sulfation, was the same as that used by Mulder and Scholtens

(1977) to block the sulfation of harmol. An additional, higher

dose of phenacetin, 500 mg/kg daily, less than half the chronic

LD 50 dose of 1.12g/kg (Boyd and Hottenroth, 1968), was used to

examine the effect of chronic dosing on the metabolism of

phenacetin.

2.5.1.2. TIME AND ROUTE

All the drugs in the respective experiments were administered

orally before midday. Pentachlorophenol was administered 45 min

prior to the phenacetin dose. Aspirin and caffeine were

administered with phenacetin.

2.5.1.3. FREQUENCY AND DURATION

With the exception of acute pentachlorophenol dosing, which was

co-administered with phenacetin on the first and seventeenth day

of treatment, all other drugs were administered daily for the

duration of the experiments.

2.5.1.4. FORMULATION

Caffeine was administered in aqueous solution. Phenacetin,

aspirin and pentachlorophenol were formulated as suspensions in

0.25% sodium carboxymethylcellulose. Aspirin and

pentachlorophenol were prepared fresh before each dose.

2.5.2.1. DRUG TREATMENTS

Five different treatments were investigated. A fresh group of

six rats was used for each investigation except in the instances

where phenacetin was co-administered with either caffeine or

aspirin. In these instances each group comprised three rats.

1. P500 : Phenacetin (500 mg/kg), administered daily for 29

days.

2. P50 : Phenacetin (50 mg/kg) administered daily for 17

days. Pentachlorophenol (100 umol/kg) administered on day 17.

3. P50/PCP : Phenacetin (50 mg/kg) co-administered with

pentachlorophenol (100 umol/kg) for one day.

4. P50/A : Phenacetin (50 mg/kg) co-administered with aspirin

(50 mg/kg) daily for 15 days.

5. P50/C : Phenacetin (50 mg/kg) co-administered with caffeine

(50 mg/kg) daily for 15 days.

2.5.2.2. URINE COLLECTION

During the 24 hr period of urine collection food was withheld

and the rats were only provided with water ad libitum. Urines

were collected at intervals of 7 days beginning from day 1,

using urine collection cages. These cages were of galvanized

iron with mesh bottoms and mounted over plastic funnels to

facilitate urine collection. The urine was allowed to run into

a measuring cylinder, immersed in a Dewar flask containing a

freezing mixture of ice and salt.

A minimal amount of distilled water was used to wash the cage

mesh bottom and the washings were allowed collect in the

receiver.

Urines and washings were collected, the volumes noted and the

urines filtered separately. Aliquots (2 ml) were removed when

required from each sample for the respective assays, which were

routinely done after each collection.

GC

I P

UPPER BAND

F 1 I

NHP 2HP

1 SPECTROPHOTOMETRY

X 580 nm

I 2H PN

LOWER BAND

1

+internal standards

ENZYMATIC HYDROLYSIS

n-glucuronidase 37°C, + arylsulfatase, 16 hr

Jr EXTRACTION

CH 20 2

'Zr METHYLATION

CF12N2 in CH3OH

Jr A

TLC

Silica gel/CHC1 3

URINE

DILUTION

H PLC

I APAP APAP-SULF APAP-GLUC

ACID HYDROLYSIS

10N Kt, 100°C, 1 hr

I PHENOXAZONE

FORMATION

pH 8-9, room temp.

I EXTRACTION

CH03

Jr CHCl3 evaporation

COLOR DEVELOPMENT

1N HO, 70°C 1

i

I

FIG. 7: Analytical scheme for the urinary metabolites of phenacetin

2.6. ANALYSIS OF THE METABOLITES OF PHENACETIN

The analytical scheme for the metabolites of phenacetin is

presented in Fig. 7.


PHENACETIN, 2-HYDROXYPHENACETIN AND 3-HYDROXYPHENACETIN

Detection and quantitation of phenacetin, 2-hydroxyphenacetin

and 3-hydroxyphenacetin was by gas chromatography (Figs. 8 and

9). Analytical procedures were essentially the same as those of

McLean et al. (1981) as modified by Veronese (1982).


PARACETAMOL AND ITS CONJUGATES

Detection and quantitation of paracetamol,and its sulfate,

glucuronyl, mercapturate and cysteinyl conjugates was by high

pressure liquid chromatography (Fig. 10). Analytical procedures

were essentially those of Rumble as cited by Veronese (1982).


2-HYDROXYPHENETIDINE

Detection and quantitation of 2-hydroxyphenetidine was carried

out spectrophotometrically. The method was the same as that

used in the hepatocyte experiments (Sec. 2.4.3.).

BLANK SAMPLE D

ET

EC

TO

R R

ES

PO

NS

E

x 258 t Ig 32 • 128

1 b ' 15 20 I 25 lb I 1 15 20

TIME min)

FIG. 8: GC trace of 2-methoxyphenacetin (upper band-TLC)

Hewlett Packard gas chromatograph 5700A; Column : SCOT-0V17 (0.45mm ID x 23.65m); col. : 205°C; inj. : 200°C; det. : 250°C; Carrier gas : 112 (55 cm/sec); FID gases : Air (20 ml/min), 112 (30 ml/min).

BAMB

I2MP

•32

BLANK SAMPLE

DE

TE

CTO

R AAMB

- "256

20

TIME (mini

‘,.

t.32 .64

6 15 1O 1'5

FIG. 9: GC trace of phenacetin (lower band-TLC)

Hewlett Packard gas chromatograph 5700A; Column : SCOT-0V17 (0.45mm ID x 23.65m); col. : 2050C; inj. : 2000C; det. : 2500C; Carrier gas : H2 (55 cm/sec); FID gases : Air (20 ml/min), 112 (30 ml/min).

BLANK SAMPLE

APAP 1

APAP-SULF

ABS

ORB

ANC

E

APAP-GLUC

1 0 0.02 1.0 0.2 0.02 0.2 TIME (min)

1 5 10 15 0 5 10

FIG. 10: HPLC ANALYSIS OF THE METABOLITES OF PHENACETIN : PARACETAMOL AND CONJUGATES OF PARACETAMOL

15

COL= :

SOLVENT :

FLOW RATE :

DETECTOR :

SENSITIVITY :

INJECTION VOLUME :

CHART SPEED :

pBondapak C18 (Waters Associates, 3.9mm ID x 30cm).

CH3CN/phosphate buffer (10mM, pH 5.0) : 3/97.

2m1/min (Waters Associates model M45 solvent delivery system).

UV:254nm (Waters Associates model 441 absorbance detector).

0.02 - 1.0 AUFS.

10p1 (Waters Associates model U6K Universal Liquid Chromatograph Injector).

15"/hr (Houston Instruments Omniscribe Recorder).

2.6.4. ASSAY FOR N-HYDROXYPHENACETIN

NHP was detected and quantitated by a modification of an earlier

method (McLean et al.,1981). An aliquot of urine (2m1) was

mixed with acetate buffer (200 ul, 1.1 M, pH 5.2), extract of

Helix pomatia (100 ul) and the internal standard, deuterated N-

hydroxyphenacetin (DNHP,10 ug in 10 ul methanol).

The urine mixture was incubated at 37 C overnight to hydrolyse

conjugated metabolites. The incubate was extracted into

dichloromethane, methylated with diazomethane and separated by

thin layer chromatography on silica gel as described before

(McLean et al.,1981). Methylated derivatives of NHP (N-

methoxyphenacetin, NMP), DNHP (deuterated N-methoxyphenacetin,

DNMP) and 2-HP (2-methoxyphenacetin, 2MP) and 3-HP (3-

methoxyphenacetin, 3-MP) migrated together (with Rf 0.26) after

two developments in chloroform. The corresponding urine zone

was eluted and NMP identified by combined gas chromatography and

mass spectrometry (GC/MS). The mass spectrum of NMP was

identical to that reported earlier (McLean et al.,1981). A

method based on multiple metastable peak monitoring (Gaskell and

Millington, 1978) after direct insertion of the sample into a

double focussing mass spectrometer, set up and operated as

described by Davies et al. (1982) in a similar assay for

warfarin, was used for quantifying NHP in rat urine.

DNMP and NMP produce significant first field free region

metastable peaks for the successive loss of the elements of the

acetyl and methoxyl groups (DNMP: m/z 213->139; NMP: m/z 209-

>135, Fig.11). These metastable peaks are not found in any of

the ring-methoxylated metabolites and so could be used to

directly measure DNMP and NMP in the presence of the other

methoxylated metabolites of phenacetin. Quantitation was by area

of the ion current versus time curve during the distillation at

120 C from the probe (Fig. 12). Blank urine gave no peaks and

the calibration curve (ratio NHP/DNHP) was linear over the range

of 0-50 ug NHP added to urine.

In urine samples collected after the administration of

pentachlorophenol on day 17 of chronic phenacetin dosing, an

interfering peak for the 209->135 reaction was encountered. This

is evident from the disparity in the distillation profiles,

shown in Fig. 12. A GC/MS analysis was carried out to determine

the source of interference. Two additional components present

in the sample were detected and tentatively identified as ethyl

hippurate and ethyl phenyl-acetyl-glycine. The molecular weight

of ethyl hippurate was 207 and it gave a large fragment in at

134, with simultaneous isotope peaks at 208 and 135.

It therefore appeared that the interference could have

originated from a 208->135 reaction, as interference peaks are

known to occur in first field free region metastable studies.

due to reactions from adjacent precursors to the same daughter

ion or reactions within fields (Lacey and Macdonald,1977).

The effect this interfering reaction has on the quantitation of

NMP could be excluded if the accelerating voltage was changed to

monitor a hypothetical 210->135 reaction, thereby allowing the

detection of a 209->135 reaction, but not a 208->135 reaction.

This was carried out and the samples were then found to have the

170

•

260

100-

80-

60-

40-

20- i f

100-

80-

60-

40-

20-

1

43

43

. 11k .

REL

ATI

VE I

NTE

NS

CH30x• NiCOCH3

11111 t

100

85

MIN .1

100

213 1

81

CH30, g NiCOCH3

0C2H5

167

1209

260

108

1351

112

139

M Z

0C 2 H5

FIG. 11: Mass spectra of N-methoxyphenacetin and deuterated N-methoxyphenacetin

Arrows show reactions monitored for quantitation of NHP.

Added 10pg NHP/2m1 urine

NMP Area: 1832

-AMP-

rea: 1717

1 0 30 60 90

Detected 9.71pg NHP/2ml urine

NMP Area: 2971

DNMP Area: 2837

STANDARD METABOLITE

0 30 60 90

TIME (sec)

FIG. 12: N-methoxyphenacetin

Profile of ion current vs. time for standard and metabolite containing urine samples.

ION

CURR

ENT

: 213+139

DNMP Area: 4119

: 213+139

DNMP Area: 339

NMP Area: 133

SAMPLE P50/PCP

SAMPLE P50/PCP ION MONITORING: 209+135

ION MONITORING: 210+135

ION

CU

RRE

NT

0 30 60 90 120 150 170

0 30 60 90

TIME (sec)

FIG. 13: N-methoxyphenacetin

Elimination of interference by increasing the accelerating voltage to ion-monitor the 210+135 reaction.

expected distillation profile, indicating the exclusion of the

interference (Fig. 13).

2.7. STATISTICAL ANALYSIS OF DATA

• A students paired t test was performed for each group of rats in

order to determine the significance of the observed changes in

the metabolism of phenacetin with the different treatments. The

change was considered significant when the value of p was less

than 0.05.

CHAPTER 3. RESULTS AND DISCUSSION

3.1. METABOLITES OF p-PHENETIDINE IN VITRO

Results of the metabolite analysis obtained from the incubation

of 0.5 mM p-phenetidine (3.4 mg in 50 ml) with freshly isolated

hepatocytes are presented in Table 1.

The expected metabolites, paracetamol, p-aminophenol,

nitrosophenetole and N-hydroxyphenetidine were not detected

although the sensitivity of the assays used provided detection

limits of 0.5 ug/ml for the compounds.

As shown in Table 1 and Fig. 14 the PN was further metabolized,

indicated by its decreasing concentration over the period of

incubation. However, the decrease in concentration was

inadequately accounted for by the formation of phenacetin and 2-

hydroxyphenetidine, the acetylated and hydroxylated metabolites

respectively. The N-acetylation of the deacetylated metabolite

of phenacetin in the rat , namely the N-acetylation of PN, is

not considered significant, because the N-acetylated metabolites

of PN are not excreted in the urine of Chester Beatty rats

gavage fed [ethyl-14-C]-p-phenetidine (Nery,1971b). However,

from the in vitro experiments performed (using freshly isolated

hepatocytes from Hooded Wistar rats) in the present work, N-

acetylatio.n of PN was significantly demonstrated. The

unaccounted for PN was probably converted to certain other

unknown metabolites or conjugates.

The relative instability of PN metabolites makes their isolation

and investigation difficult. Nevertheless, the tendency of

isolated hepatocytes to hydroxylate and acetylate the xenobiotic

suggested the elimination of PN via these pathways and explained

in some measure the increased 2HPN formed in vivo by inhibition

of sulfation or stimulation of aromatic hydroxylation (Sec.

3 .2.2.).

Table 1: Metabolism of p-phenetidine in isolated rat hepatocytes

CONCENTRATION FOUND IN SUPERNATANT (ug/m1)

CONTROL (without hepatocytes)

Sample Time No. (min)

p-phenetidine

1 0 64.35

2 30 -

3 60 61.58

4 90 -

5 120 60.81

HEPATOCYTE INCUBATE

p-phenetidine phenacetin 2-hydroxy-p-phenetidine

52.78 2.08 -

51.62 3.44 -

49.36 4.17 0.58

40.87 3.67 0.70

34.79 3.26 0.88

1. Typical of 4 experiments.

2. Paracetamol, p-aminophenol, p-nitrosophenetole and N-hydroxyphenetidine were not detected.

CONCENTRATION (pg/m1)

t•J "C) 0 'CI 0- 01

• (D "0 kC rt CD-0 (D ✓ CD 0 0 O (D (D X rt rt

• w • I CD

CD CD

.0 O M

H. CD ✓t

CD- CD H.

CD

rt

•

0

CD

rr

rt 0 CD

rr CD

• • ■ •

\/

•

oo J -A%

• o -

0 M

STIO

CIE

39N

h eneti d

ine i n

i sol at ed

rat he

CONCENTRATION (pg/m1) Li' 0N --,

o o o o o I 1 I I I

Ar ■

3.2. METABOLITES OF PHENACETIN IN VIVO

Buch et al. (1967) demonstrated that the major fraction (58%) of

metabolized phenacetin, in the rat, is accounted for as

paracetamol and its sulfate and. glucuronide conjugates. From

the results presented in Table 2 and the graphic

representations, Figs. 15 to 21, it is seen that a major

fraction of the phenacetin dose was deethylated to paracetamol

which was excreted in urine either as free paracetamol or as

conjugated paracetamol-sulfate and paracetamol-glucuronide. This

finding was compatible with the findings of earlier in vivo

studies (Brodie and Axelrod,1949; Nery 1971b). The cysteinyl

and mercapturate conjugates reportedly found in albino rats of

the Birmingham Wistar strain, when treated with phenacetin

(Smith and Griffiths,1976) were not detected in the Hooded

Wistar strain of rats used in these experiments.

Paracetamol-sulfate (APAP-SULF) was the major metabolite found

in the present experiments, while paracetamol-glucuronide (APAP-

GLUC) levels were higher than those reported by Buch et al.

(1967).

Hydroxylation of phenacetin was in evidence in the present work.

N-hydroxyphenacetin (NHP) was found present up to 0.3% of the

dose of phenacetin. 2-Hydroxyphenacetin (2HP), measured as

0.07% of the dose of phenacetin in other earlier studies was

present upto a maximum of 0.06% of the dose of phenacetin in the

present study. 3-Hydroxyphenacetin (3HP) was not detected. 2-

Hydroxyphenetidine (2HPN) was also formed to the maximum extent

of 0.8% of the dose of phenacetin as compared to 6% of the

phenacetin dose reported as the sulfate conjugate of 2HPN (Buch

et al.,1967). The N—deacetylated metabolite 2HPN comprised only

a minor fraction of the administered dose of phenacetin, an

observation which has been similarly demonstrated in earlier

experiments (Brodie and Axelrod,1949; Smith and Williams,1949),

further indicating the metabolically inert nature of the acetyl

group of phenacetin as previously suggested by Nery (1971b).

Unchanged phenacetin was found up to 0.8% of the dose

administered.

Table 2: Metabolites of phenacetin in the rat with different treatments

Days TREATMENT

PERCENTAGE DOSE OF METABOLITES OF PHENACETIN

APAP APAP-SULF APAP-GLUC NHP 2HP 2HPN P TOTAL

1 4.05 61.03 22.57 0.046 0.00027 0.779 0.002 88.48 (0.31) (2.50) (0.91) (0.002) (0.00002) (0.060) (0.000)

8 5.98 58.95 35.44 0.120 0.00040 0.516 0.011 101.02 P500 (0.31) (7.48) (4.35) (0.014) (0.00003) (0.031) (0.001)

15 6.64 52.99 49.76 0.243 0.00085 0.614 0.005 110.25 (0.61) (0.88) (3.74) (0.020) (0.0001) (0.066) (0.000)

29 6.98 48.51 55.19 0.314 0.00078 0.750 0.003 111.75 (0.61) (3.42) (4.99) (0.021) (0.00007) (0.118) (0.000)

, 2.72 76.27 13.20 0.102 0.043 0.016 0.145 92.49 (0.16) (1.70) (1.11) (0.014) (0.002) (0.001) (0.009)

1 2.34 71.97 \ 15.95 0.111 0.043 0.018 0.374 90.81 (0.28) (2.24) (0.96) (0.010) (0.011) (0.001) (0.027)

8 P50 4.13 88.72 15.07 0.099 0.028 0.007 0.392 108.45 (0.29) (1.61) , (1.05) (0.014) (0.002) (0.000) (0.118)

15 5.14 102.29 15.31 0.144 0.038 0.012 0.306 123.28 (0.27) (6.77) (1.84) (0.026) (0.005) (0.000) (0.053)

17* 4.43 75.32 15.33 0.123 0.035 0.018 0.808 96.06 (0.37) (8.89) (1.23) (0.013) (0.012) (0.001) (0.058)

Table 2: (continued)

Days TREATMENT

PERCENTAGE DOSE OF METABOLITES OF PHENACETIN

APAP APAP-SULF APAP-GLUC NHP 2HP 21IPN P TOTAL

1 3.62 78.7 13.55 0.103 0.038 0.123 0.116 96.25 (0.57) (1.33) (1.33) (0.007) (0.006) (0.009) (0.022)

8 P50/A 4.78 74.5 17.74 0.214 0.054 0.170 0.115 97.57 1 (0.04) (5.64) (1.11) (0.054) (0.004) (0.014) (0.026)

15 4.77 77.3 16.21 0.221 0.048 0.174 0.090 98.72 (0.50) (3.05) (0.80) (0.027) (0.002) (0.001) (0.010)

1 3.92 80.22 13.03 0.115 0.059 0.160 0.158 97.66 (0.68) (1.53) (0.17) (0.009) (0.017) (0.010) (0.010)

8 P50/C 4.40 65.91 15.04 0.132 0.039 0.197 0.109 85.83 (0.57) (4.91) (0.82) (0.023) (0.006) (0.022) (0.006)

15 4.80 73.33 13.54 0.189 0.058 0.235 0.113 92.26

(0.33) (12.06) (1.45) (0.029) (0.012) (0.003) (0.014)

(*) pentachlorophenol (100 umol/kg) adM)inistered.

Each value is the mean (± SE) of 6 rats, except for P50/A and P50/C where 3 rats were used.

P500

100- 100-

80- 80- me

60 - • ** 60-

• 40- 40 -j

20- 20-

s s 1 8 15 22 29 1

P50/A 100 100

80- • • • 80- • 60- 60-

40- 40 -

20- 20-

PERC

ENTA

GE

DOS

E

8

15 17

P50/C

P50

1 8 15 1 8 15

TIME (DAYS)

FIG. 15: Paracetamol sulfate

Profile of the percentage of phenacetin dose vs. time

P500 : Phenacetin (500mg/kg) daily.

P50 : Phenacetin (50mg/kg) daily, : (A) with pentachlorophenol (100pmol/kg) on day 17, : (0) with pentachlorophenol (100pmol/kg) for one day

(another group of rats was used for this point only).

P50/A : Phenacetin (50mg/kg) and aspirin (50mg/kg) daily.

P50/C : Phenacetin (50mg/kg) and caffeine (10mg/kg) daily.

(*) p < 0.05; (**) p < 0.01 versus day 1.

60-

40-

20-

1 I t

1 8 15 17 I

1 8 15 22 29

** 30- •

20-

• •- A •

•

10 -

P500

P50

PERC

ENTA

GE D

OSE

P50 /C

• • •

P50/A

30- 30-

20- 20-

•

10 - 1 0-

1 8 15 1 8 15

TIME (DAYS)

FIG. 16: Paracetamol-glucuronide

Profile of the percentage of phenacetin dose vs. time.

Refer to the legend of Fig. 15 for particulars of treatments.

PERC

ENTA

GE

DO

SE

• 00 —

00

—

00 —

• -

Lii

N.)

U.)

0

0

0

0

a

•

TOME39DPIEd

•*

ts.)

LA

) 0

0

0

0

0

(sxva) aka'

Prof ile of the percentage of phenacetin d ose vs .

Refer to the legend of Fig . 15 for particulars of treatments .

•

.04-

P50

.05-

** • .04-

.03-

.02-

.01-

22 29 1 8 15 17

P50/C

.06-o

•

.04-

.02-

P500

.02-

.0010-

.0008-

.0006-

.0004-

.0002- •

1 8 15

PERC

ENTA

GE D

OSE

P50/A

.06-

1 1 8 15 1 8 15

TIME (DAYS)

FIG. 18: 2-hydroxyphenacetin



** •

0.3- 0.3-

0.2- 0.2-

0. 1- 0.1-

1 8 15 22 29 1

P50/A PERC

ENTA

GE

DO

SE

8 15 17

P50/C 0.3-

0.2

0.3

0.2-

• • 0.1 0. 1

P500 P50

•

8

15 1 8 15

TIME (DAYS)

FIG. 19: N-hydroxyphenacetin



P500

.03-

0.8- * .02 • • • 0.6- * *

• 0.4 - .01-

0.2

1 8 15 22 29 1

P50/A

.3- .3-

.2 .2

• • •

.1 .1-

P50

PER

CEN

TAG

E DO

SE

I 8

15 17

P50/C

1

8

15 1 8 15

TIME (DAYS)

FIG. 20: 2-hydroxyphenetidine



1.0

0.8-

0.6-

0.4-

P50

* * •

• • •

0.2- •

1 8 15 17

P50/ C

0.5-,

0.4 -

0.3-

0.2- •

0.1 - • •

.010-

.008 -

.006-

.004-

0.4-

0.3-

0.2-

0. 1-

P500

* * •

•

1 8 15 22 29

P50/A

•

1

8 15

1 8 15

TIME (DAYS)

FIG. 21: Phenacetin



3.2.1. P500 TREATMENT

In rats dosed with 500 mg/kg of phenacetin daily, between 48%

and 61% of the dose was excreted as APAP-SULF (Fig. 15). The

levels of APAP-SULF in the present work were higher than those

reported by Smith and Timbrell (1974) and declined over the 29-

day period of study. A progressive depletion, with time, of

cytosolic sulfotransferase sulfation was therefore inferred.

The progressive decrease in sulfation was compensated for with a

simultaneous increase in glucuronidation. Markedly increased

levels of APAP-GLUC were witnessed during the experimental

period (Fig. 16). The fraction of dose excreted as APAP-GLUC

increased from 23 % to 55 % during the dosing period, higher

than the maximum of 18 % of the dose reported previously (Smith

and . Timbrell, 1974)

The glucuronidation of phenacetin with UDP-glucuronyltransferase

and its sulfation with cytosolic sulfotransferase enzymes of

phenace tin are competitive pathways of metabolism (Mulder and

Meerman,1978). Their precedence in the conjugation process are

inversely related, as demonstrated by Kadlubar et al. (1980),

Meerman et al. (1980) and Mulder and Scholtens (1977) in studies

on the regulatory role of sulfation in some biological

processes. The results of the present work were consistent with

this inverse relationship and it was seen that glucuronidated

levels of the drug were notably increased when sulfation was

suppressed or impaired.

Smith and Timbrell (1974) found that free APAP is excreted to

the extent of 5% of the dose of phenacetin. In the present

study the fraction of unconjugated APAP increased from 4% to 7%

of the dose of phenacetin, over the experimental period (Fig.

17). This indicated that not all of the decreased sulfation was

compensated for by an increase in glucuronidation alone.

Smith and Timbrell (1974) found 2HP present to the extent of

3.0% of the dose of phenacetin, but did not measure the

metabolites NHP and 2HPN in rat urine. NHP and 2HPN are

significantly relevant to the toxicity of phenacetin. The

significant increase observed in the formation of 2HP and NHP

over the experimental period (Figs. 18,19) could be a result of

induction of the hydroxylation pathway. Auto-induction of 2HP

formation was obvious in the first fortnight but did not

increase further in the next. This resulted in its continued

high level of formation. The induction of formation of NHP

continued for the entire 29-day period of study and was

evidenced by the increasingly higher levels of NHP formed.

NHP had first been reported as an in vivo metabolite in the rat

by McLean et al. (1981). The arylhydroxamic acids (Miller and

Miller,1966a ; Weisburger and Weisburger,1973) including NHP

(Calder et al.,1976) are known to be carcinogenic. The toxicity

of phenacetin seen on chronic administration could therefore be

due to the increased formation of NHP.

However, irrespective of the reason for the increased

hydroxylated products formed, the increased levels of NHP seen

on administration of the 500 mg/kg dose daily, could well be a

reason for the chronically induced toxicity of phenacetin.

The amount of deacetylated metabolites excreted in the urine of

rats treated with phenacetin is increased when larger doses of

phenacetin are administered (Raaflaub and Dubach,1969). A

higher percentage of the dose of phenacetin present as 2HPN was

seen on this treatment (P500) in comparison with that seen at

the lower dose of phenacetin treatments (P50; P50/Ai P50/C).

Formation of 2HPN exhibited a decreasing trend from 0.8% to 0.5%

of the phenacetin dose (Fig. 20). This decrease was most

prominent in the first week and was ostensibly related to the

precedence of the glucuronidation pathway and the increased

direct hydroxylation of phenacetin to 2HP and NHP. An increase

or return to normality after the first week was seen in which

2HPN reached a maximum of 0.75% of the dose on day 29 (Table 2).

The percentage of the dose of phenacetin found unchanged in

urine increased in the first week from 0.002% to 0.011% and then

decreased over the next three weeks (Fig. 21). It appeared

logical to assume that the kinetics of metabolism permitted the

accumulation of phenacetin until steady state had been attained.

Subsequently induced metabolism converted more free phenacetin

to its respective metabolites.

3.2.2. P50 TREATMENT

For rats on this dosage regimen a larger percentage of the dose

was excreted as APAP-SULF, in comparison to the P500 treated

rats. A significant increase in APAP-SULF formation was

evidenced over the 15 day experimental period (Fig. 15). The

sulfation pathway coped with this dose of phenacetin and its

induction resulted in increased sulfation. Pentachlorophenol

(PCP, 100uM)) a known inhibitor of sulfation (Mulder and

Meerman,1978 ; Meerman et al.,1980) given orally on day 1 of the

experiment did not alter the level of APAP-SULF in rats

significantly (Fig. 15), contrary to expectations held on the

basis of the earlier experiments in acutely dosed rats (Mulder

and Meerman,1978)- Administered on day 17 of the study, PCP

inhibited sulfation significantly (p<0.05) though incompletely

(Fig. 15). The inhibition of sulfation was 27%, compared to

100% found by Mulder and Scholtens (1977) in their experiments

using the same dose of PCP intraperitoneally. Therefore, PCP

administered orally exerts only partial sulfatioti inhibition in

rats treated chronically with phenacetin.

The APAP-GLUC levels were much lower in comparison to the P500

treated rats and remained unaltered at 15% of the phenacetin

dose (Fig. 16) during the course of the experiment. This was

not in accordance with the expectation that the glucuronidation

activity would be reduced as a consequence of the induction of

the competitive sulfation pathway (Mulder and Meerman, 1978).

Contrary to expectations therefore, the progressive increase in

sulfation proceeded with no apparent effect on glucuronidation

at this dose. Mulder and Scholtens (1977) have demonstrated the

suppression of glucuronidation with PCP (100uM, IP) in the rat.

In the present work PCP (100uM, po) did not affect the

glucuronidation of APAP. The percentage of the dose of

phenacetin excreted as APAP-GLUC remained unaffected, thereby

further establishing the selective sulfation inhibitory effect

of PCP.

The percentage of dose excreted as APAP was lower than that seen

in the P500 treated animals. The APAP fraction increased from

2.3% to 5.1% of the dose of phenacetin over the study period

(Fig. 17). PCP did not alter the percentage of the dose of

phenacetin excreted as APAP when given on day 1, but _-

significantly (p<0.05) lowered it when co-administered on day 17

of the chronic treatment with phenacetin (Fig. 17).

A marked decrease in 211P formation in the first week was

followed by a recovery in the next (Fig. 18). PCP did not alter

the percentage dose of phenacetin converted to 2HP when given on

day 1 but lowered it (p<0.05) when co-administered with

phenacetin on day 17 (Fig. 18).

Formation of NHP was marginally decreased in the first week and

then significantly increased in the next (Fig. 19). Induction

of the N-hydroxylating pathway was noticed in the second week of

the experiment.

The levels of NHP were not affected on day 1, but were lowered

(p<0.05) on day 17 by the co-administration of PCP and

phenacetin. PCP therefore, apparently inhibited the ter

hydroxylation of phenacetin to NHP and 2HP in rats treated

chronically with phenacetin.

The percentage of dose excreted as 2HPN diminished sharply in

the first week and partially recovered in the second week of the

experiment (Fig. 20). PCP did not affect the formation of 2HPN

significantly on day 1, but caused a steep rise in 2HPN levels

on day 17 of the experiment when co-administered with phenacetin

(Fig. 20).

The partial block of sulfation by PCP resulted in an increased

formation of 2HPN. This suggested a metabolic shift towards

increased indirect hydroxylation of phenacetin via PN or

increased deacetylatcon of 2HP or both. Phenacetin excretion

was also increased. However, the total increase in excretion of

2HPN and phenacetin was less than 1 % of the dose of phenacetin

administered. This did not account for the large decrease (27 %

of dose) in sulfation caused by PCP on day 17.

3.2.3. P50/A TREATMENT

Aspirin has been shown to be nephrotoxic (Prescott,1970; Nanra

and Kincaid-Smith,1972b; 1973b) but its possible potentiation of

phenacetin-induced carcinogenicity and nephrotoxicity require

further investigation. Altered metabolism of phenacetin

resulting from an aspirin-phenacetin (Thomas et al.,1973 ; 1974)

or paracetamol-phenacetin (Whitehouse et al.,1977) metabolic

interaction have been reported earlier • In the present instance

when aspirin and phenacetin were co-administered the percentage

dose of phenacetin excreted as APAP-SULF showed no increase over

the 15 day period (Fig. 15) as compared to the increased levels

seen on the administration of the same dose of phenacetin alone.

The induction of sulfation was interfered with and as a

consequence APAP-SULF levels were marginally depressed in the

first week and only partially recovered in the second week.

These results were in accordance with those of Thomas et al.

(1974) for phenacetin-aspirin co-administered to rats,

Whitehouse et al. (1977) for paracetamol-aspirin co-administered

to mice and Wong et al. (1976) for paracetamol-aspirin co-

administered to hamsters. In these studies the significant

metabolic effect of aspirin is the reduction of APAP sulfation.

The suppression of sulfation accounted for the increased

compensatory glucuronidation which yielded higher levels of

APAP-GLUC in the first week and subsequently slightly lower

elevated levels (Fig. 16). An increase in glucuronidation was

also indicative of the enhanced UDP-glucuronyltransferase

activity that salicylates are reported to cause (Hanninen and

Aitio,1968; Diamond et al.,1982).

Free APAP levels increased from 3.6% to 4.8% of the phenacetin

dose during the experiment (Fig. 17), a trend similar to that

observed in the P50 treated rats.

The hydroxylation of phenacetin to 2HP was increased in the

first week and lowered marginally in the next (Fig. 18). The N-

hydroxylated product NHP was increasingly formed in the first

week of treatment and continued a gradual escalation in the next

week (Fig. 19). The 2HPN fraction increased over the 15 day

period, most of the increase occurred in the first week of the

experiment.

The co-administration of phenacetin and aspirin therefore

resulted in increased aromatic- and N-hydroxylation. This could

be a result of the direct induction of the hydroxylation pathway

or a consequence of the partial sulfation inhibition. Analgesic

mixtures containing aspirin in combination with phenacetin are

known to be more nephrotoxic than analgesic mixtures in which

aspirin is excluded or is replaced with phenazone (Nanra et al.,

1980). Johansson (1981) explains the occurrence of renal pelvic

tumors (only seen in rats treated with phenacetin or phenazone

alone or in combination with caffeine) as a consequence of the

altered metabolism of phenacetin, increasing the production of

NHP, a postulated liver carcinogen. The induced N-hydroxylation

of phenacetin by aspirin was therefore a noteworthy observation

of significant interest in the context of potentiated

phenacetin-induced carcinogenicity and nephrotoxicity.

The unchanged phenacetin levels declined over the period of

study (Fig. 21). The decline was mainly seen in the second week.

3.2.4. P50/C TREATMENT

Caffeine continues to be included in analgesic mixtures today.

The contribution of caffeine to phenacetin-induced toxicity

needs to be investigated. Research into the mutagenic activity

and carcinogenic character of caffeine has indicated its

mutagenic activity in lower organisms and its association with

bladder cancer (Kuhlmann et al.,1968; Cole,1971), while other

investigations have found that caffeine seemed to decrease the

risk of malignancy in rats treated with phenacetin (Granberg-

Ohman et al.,1980).

In the present work, when phenacetin and caffeine were co-

administered a trend similar to to that observed in the P50/A

treated rats was witnessed. The APAP-SULF levels showed no

increase over the 15 day period of study (Fig. 15), quite unlike

the increased levels of APAP-SULF seen when the same dose of

phenacetin was administered alone.

The induction of sulfation was inhibited to a degree by caffeine

and consequently conversion of phenacetin to APAP-SULF in the

first week was marginally depressed and only fractionally

recovered in the second week.

Increased glucuronidation, apparent in the first week,

compensated for the partial inhibition of-sulfation (Fig. 16).

Free APAP levels increased from 3.9% to 4.8% of the phenacetin

dose over the duration of the experiment (Fig. 17). This

observation was common to the P50 and P50/A treated rats.

The hydroxylation of phenacetin to 2HP decreased in the first

week and then recovered in the next (Fig. 18) an observation

similar to that seen in the P50 treated rats. Thus caffeine did

not affect the formation of 2HP.

A progressive increase in the percentage dose of NHP was

observed over the 15 day experiment (Fig. 19). The increase was

more evident in the second week. Contribution to induction of

N-hydroxylation by caffeine was only marginal, as compared to

that of aspirin when co-administered with phenacetin. However,

it still was significantly relevant to the potentiation of

phenacetin-induced toxicity.

An increase in the percentage dose of 2HPN occurred over the

entire 15 day period of study (Fig. 20). Caffeine therefore

noticeably induced the hydroxylation of phenacetin to 2HPN. The

unchanged phenacetin levels remained unaltered during the period

of study (Fig. 21).

CONCLUSION

Sulfation plays an important role in the metabolism of

phenacetin in the rat. Most of the drug was accounted for as

the major metabolite, paracetamol-sulfate. Partial suppression

of the sulfation pathway with pentachlorophenol increased only

the fraction of phenacetin metabolized to 2-hydroxyphenetidine.

It did not increase the formation of 2-hydroxyphenacetin or N-

hydroxyphenacetin.

As a result of sulfation inhibition, more phenacetin was

excreted unchanged. The phenacetin metabolized via deethylation

and the sulfation pathway was not alternatively accounted for

through any other metabolic pathway. This may indicate a limited

capacity of the alternative pathways of phenacetin metabolism in

the rat.

Autoinduction of hydroxylating enzymes was seen at both high and

low doses of phenacetin used, though to a lesser degree with the

lower dose. Induction of N-hydroxylation was enhanced by aspirin

and caffeine. This significant observation could explain the

potentiated toxicity encountered in the chronic abuse of

aspirin-phenacetin-caffeine combinations. The increase in N-

hydroxylation was more likely a result of direct stimulation of

the hydroxylating enzymes rather than an effect of the

accompanying partial suppression of sulfation resulting when

these drugs are co-administered with phenacetin.

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Metabolism of phenacetin in the rat - University of …SouzaConradAgnello1984... · METABOLISM OF...

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